A. Field of the Invention
The present invention relates to a method of performing a sandwich type immunoassay for the detection of an antibody using a coated antigen and a labeled antigen, wherein the labeled antigen and the coated antigen are produced from genetically unrelated organisms ("heterologous species"). More particularly, the present invention relates to a solid phase method and device for detecting antibodies to some antigen ("X") in mammalian fluids or tissue wherein the antibody of interest is sandwiched between an antigen "X". which is bound to a solid phase, and an antigen "X" or a common epitope thereof, which is derived from a heterologous organism and bound to a label. The method and device of the present invention are useful in laboratory medicine because they permit a physician or veterinarian to determine rapidly and specifically whether a patient has an immune response to a particular organism or antigen, thereby reflecting present or prior exposure to that organism or antigen. In particular, the method and device of this invention is useful in rapidly and specifically detecting an immune response and thus exposure to the HTLV III virus which has been associated with acquired immune deficiency syndrome (AIDS).
B. Prior Art
Most immunoassays for detection of an antibody involve an antigen coated solid phase which is used to capture antibodies specific to the coated antigen. The captured antibodies are then quantitated and/or identified using an anti-antibody to which a detection system such as an enzymes or radioisotope is conjugated.
A second type of immunoassay also utilizes an antigen coated solid phase to capture antigen specific antibodies. However, unlike the first immunoassay above, the captured antibodies of the second type of immunoassay are quantitated and/or identified using an antigen cross-linked to a radiolabel or an enzyme which serves as a detection system. In this second type of immunoassay, both the antigen coating the bead and the antigen bearing the label are identical and from the same organism (source) and bear the same contaminants. Hereinafter, antigens which are obtained from the same or related species of organisms are referred to as "homologous" antigens.
For example, current immunoassays for the detection of antibodies to HTLV III (anti-HTLV III) require extensive specimen dilution; utilize virus purified from human cell line H9 to coat a solid phase; and finally, utilize a non-specific probe (e.g. Anti-human IgG, conjugated to horseradish peroxidase (HRPO)). In these immunoassays, biological fluids are screened for the presence of anti-HTLV III by contacting the unknown sample with a disrupted virus coated solid phase. Antibody to the HTLV III virus then binds to the virus coated solid phase. Because the disrupted virus produces a plurality of antigens, which are coated to the solid phase, any antibody in the sample, which is reactive against contaminants in the disrupted viral preparations, will also bind to the coated solid phase. Once antibody is bound to the solid phase, it will produce a significant number of "false positives" regardless of whether a labeled antibody (first type immunoassay) or a labeled homologous antigen (second type immunoassay) is used as the probe.
Accordingly, it is an object of the present invention to provide an immunoassay method and device for detecting and/or quantitating anti-HTLV III which has specificity, i.e., overcomes this "false positives" problem.
Unlike the immunoassays, competitive binding assays exist which overcome some of the specificity problems. For example, the competitive protein binding assay for anti-HTLV III uses recombinant antigens and allows discrimination between antibodies against HTLV III envelope (ENV) and HTLV III Core antigens. However, this procedure is lengthy, typically being carried out overnight. Moreover, it requires two solid phases i.e., beads coated with p41 envelope antigen and beads coated with p24 core antigen.
Accordingly, it is an object of the present invention to provide an immunoassay for anti-HTLV III which is not only rapid but which also has the specificity of a competitive binding assay without requiring two solid phases. cl SUMMARY OF THE INVENTION
The present invention is directed to an improvement in the method of performing a sandwich immunoassay for detecting antibody to a specific antigen in a test sample wherein a first antigen specific to the antibody to be detected is immobilized on a solid phase, wherein the antibody to be detected in the test sample binds to the first antigen thereby becoming immobilized, wherein the immobilized antibody further binds a second antigen bearing a label, and wherein the first antigen and the second antigen are derived from a homologous source, the improvement comprising deriving the second antigen from a source that is heterologous to the source of the first antigen.
In particular, the present invention is directed to a method for detecting an antigen specific antibody in a test sample comprising the steps of:
(a) immobilizing a first recombinant derived antigen specific to the antibody to be detected on a solid phase;
(b) contacting the solid phase produced in step (a) with an aqueous phase test sample containing or suspected of containing the antigen specific antibody;
(c) contacting the solid phase produced in step (b) with an aqueous phase containing a second recombinant derived antigen having a label affixed thereto, the second recombinant derived antigen being derived from a source that is heterologous to the source of said first recombinant derived antigen;
(d) separating the aqueous phase from the solid phase;
(e) measuring the presence of the label on the solid phase or in the liquid phase to detect and/or titer the presence of antibody in the test sample.
This invention further relates to a modification of the described device and method wherein both the coated antigen and the labeled antigen, which are from heterologous sources, need not be identical so long as the antigens have at least one antigenic determinant ("epitope") in common.